Autophagy Beclin 1-independent autophagy

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Beclin 1-independent autophagy contributes to apoptosis in cortical neurons Yulia Grishchuk, Vanessa Ginet, Anita C. Truttmann, Peter G.H. Clarke & Julien Puyal Published online: 01 Oct 2011.

To cite this article: Yulia Grishchuk, Vanessa Ginet, Anita C. Truttmann, Peter G.H. Clarke & Julien Puyal (2011) Beclin 1-independent autophagy contributes to apoptosis in cortical neurons, Autophagy, 7:10, 1115-1131, DOI: 10.4161/ auto.7.10.16608 To link to this article: http://dx.doi.org/10.4161/auto.7.10.16608

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Basic Research Paper

Basic Research Paper

Autophagy 7:10, 1115-1131; October 2011; © 2011 Landes Bioscience

Beclin 1-independent autophagy contributes to apoptosis in cortical neurons Yulia Grishchuk,1,† Vanessa Ginet,1,2,† Anita C. Truttmann,2 Peter G.H. Clarke1 and Julien Puyal1,* Département de Biologie Cellulaire et de Morphologie; Université de Lausanne; Lausanne; Switzerland; 2Division de Néonatologie; Département Médico—Chirurgical de Pédiatrie; Centre Hospitalier Universitaire Vaudois Lausanne; Switzerland

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These authors contributed equally to this work.

†

Key words: autophagy, apoptosis, neurons, cell death, lysosomes, caspases

Downloaded by [82.78.191.159] at 08:50 02 July 2015

Abbreviations: AcHex, β-N-acetylhexosaminidase; AIF, apoptosis-inducing factor; AP, acid phosphatase; ANOVA, analysis of variance; BA1, bafilomycin A1; LAMP1, lysosomal-associated membrane protein 1; LC3, microtubule-associated protein 1 light chain 3; 3-MA, 3-methyladenine; Q-VD-OPH, quinoline-Val-Asp(ome)-Ch2-O-phenoxy; PtdIns3k, phosphatidylinositol-3kinase; STS, staurosporine

© 2011 Landes Bioscience. Neuronal autophagy is enhanced in many neurological conditions, such as cerebral ischemia and traumatic brain injury, but its role in associated neuronal death is controversial, especially under conditions of apoptosis. We therefore investigated the role of autophagy in the apoptosis of primary cortical neurons treated with the widely used and potent pro-apoptotic agent, staurosporine (STS). Even before apoptosis, STS enhanced autophagic flux, as shown by increases in autophagosomal (LC3-II level, LC3 punctate labeling) and lysosomal (cathepsin D, LAMP1, acid phosphatase, β-hexasominidase) markers. Inhibition of autophagy by 3-methyladenine, or by lentivirally-delivered shRNAs against Atg5 and Atg7, strongly reduced the STS-induced activation of caspase-3 and nuclear translocation of AIF, and gave partial protection against neuronal death. Pan-caspase inhibition with Q-VD-OPH likewise protected partially against neuronal death, but failed to affect autophagy. Combined inhibition of both autophagy and caspases gave strong synergistic neuroprotection. The autophagy contributing to apoptosis was Beclin 1-independent, as shown by the fact that Beclin 1 knockdown failed to reduce it but efficiently reduced rapamycin-induced autophagy. Moreover the Beclin 1 knockdown sensitized neurons to STS-induced apoptosis, indicating a cytoprotective role of Beclin 1 in cortical neurons. Caspase-3 activation and pyknosis induced by two other pro-apoptotic stimuli, MK801 and etoposide, were likewise found to be associated with Beclin 1-independent autophagy and reduced by the knockdown of Atg7 but not Beclin 1. In conclusion, Beclin 1-independent autophagy is an important contributor to both the caspase-dependent and -independent components of neuronal apoptosis and may be considered as an important therapeutic target in neural conditions involving apoptosis.

Do not distribute. Introduction

Enhanced autophagy has been implicated in various neurological conditions including cerebral ischemia and head injury,1,2 but its role is controversial, and there is evidence that it can promote either cell survival or cell death according to the situation.3,4 Early arguments for a death-promoting role focused on morphological descriptions of dying cells during development, when autolysosomes can fill the cytoplasm and digest nuclear DNA.5 Subsequent evidence in favor of this interpretation came from numerous reports that “autophagic cell death” (i.e., cell death manifesting intense autophagy) can be prevented by pharmacological prevention of autophagy6-8 or by knockdown of autophagy genes.9-12 On the basis of such evidence, most researchers accept that autophagy is a major mediator of cell death in a wide

range of important situations,13 including normal development and metamorphosis,5,14 AIDS,15 cerebral ischemia1,2 and cancer therapy.16 There is nevertheless controversy, and some opponents of this view minimize the role of autophagy in cell death.17 Nevertheless, a major remaining question is whether autophagy does so by a separate death effector mechanism independent of apoptosis, or whether it is a trigger for the latter or dependent upon it. There is evidence that both alternatives may be true depending on the situations.13 In support of an independent death mechanism, autophagy has been shown in some cases to promote caspase-independent cell death9,10,18 and an alternative death pathway in cells incompetent for apoptosis.11,12 On the other hand, there is evidence that autophagic pathways can modulate caspasemediated apoptosis.2,19-21 Furthermore, it is now well demonstrated that autophagy and apoptosis can share common regulators.4,13,22

*Correspondence to: Julien Puyal; Email: [email protected] Submitted: 12/22/10; Revised: 05/20/11; Accepted: 05/24/11 http://dx.doi.org/10.4161/auto.7.10.16608 www.landesbioscience.com Autophagy

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It is therefore important to clarify the relationship between autophagy and apoptosis as a prelude to neuroprotection by interfering with autophagy.1,2,20,23,24 We therefore here investigate the role of autophagy in neuronal apoptosis caused by classical apoptotic stimuli, especially the protein kinase inhibitor staurosporine.25,26


Results A strong autophagosomal increase precedes STS-induced apoptosis in cortical neurons. Staurosporine (STS), a classical apoptotic stimulus that has been previously widely characterized to induce apoptosis in several different cell types including primary neuronal cultures,25-27 was used to promote apoptosis in primary cultures of cortical neurons. In the present experiments, 1 μM STS induced neuronal death as demonstrated by a progressive increase in the number of pyknotic nuclei from 6 h of STS treatment (Fig. 1A) or in lactate dehydrogenase (LDH) release into the medium (Fig. S1a). As expected, STS gradually but strongly increased caspase-3 activation as shown by immunoblotting for cleaved caspase-3 (Fig. 1B and Fig. S1b), and also for α-fodrin, which showed an increase in the 120 kDa caspase-dependent cleavage fragment from 6 h of STS (Fig. S1c). Moreover the 145/150 kDa calpain-dependent cleavage fragment of α-fodrin likewise increased, indicating progressive calpain activation (Fig. S1c). Interestingly, along with the induction of caspase-3 and calpains, STS-treated neurons showed raised levels of autophagosomal marker LC3-II. LC3-II detected by immunoblotting was increased as early as 30 min after STS exposure, reaching the maximum value at about 12 h and decreasing by 24 h, suggesting a substantial increase in autophagosome formation after STS treatment (Fig. 1B and Fig. S1b). Together, the pattern of cleaved caspase-3 and LC3-II expressions indicates that autophagosome enhancement (30 min–12 h) is followed by a progressive activation of caspase-3 (1 h–24 h). Additionally, an increase in autophagosome formation over time was demonstrated by time-lapse imaging in neurons overexpressing sapphire-LC3 (Fig. S1d) or by counting the number of LC3-positive dots per mm2 and per neuron following immunolabeling against LC3 (Fig. 1C). Both results show a strong and gradual increase in LC3-positive vesicular structures as early as 3 h after STS. Lysosomal activation and autophagic flux are induced in STS-induced neuronal apoptosis. To demonstrate that STSinduced autophagosome increase was caused by activation of the autophagy/lysosomal pathway and was not a result of impaired downstream autophagosome fusion and degradation by lysosomes, we investigated the effect of STS on lysosomal activity. An enzymatic assay for acid phosphatase (AP) was performed on protein extracts of cortical neurons treated with STS for different times (Fig. 2A). AP activity was moderately increased after 3 h and 6 h of STS compared with the basal activity of control neurons, but then returned to control levels at 12 h and 24 h. But in contrast to this minor change in the total AP activity, we found by AP histochemistry a most striking change in the distribution of the activity, which appeared in a greater number of large

puncta in the 6 h STS-treated neurons, as shown in Figure 2B. In fact, the percentage of small puncta (≤0.3 μm2) per neuron was not affected, but there was a switch from intermediate-(>0.3 and ≤0.9 μm2) to large-(>0.9 μm2) size puncta in STS-treated neurons, reflecting putatively the creation of autolysosomes by the fusion of autophagosomes with lysosomes. Cytochemical staining for another lysosomal enzyme, β-N-acetylhexosaminidase (AcHex), was also performed and clearly showed a strong increase in AcHex activity in larger puncta following 6 h of STS compared with control neurons (Fig. 2C). Immunocytochemical staining for two lysosomal markers, lysosomal-associated membrane protein 1 (Lamp1) and the lysosomal protease cathepsin D, confirmed the enlargement of lysosomes 6 h after STS exposure (Fig. S1e). Double immunolabeling for Lamp1 and LC3 showed LC3-positive puncta (autophagosomes) and enlarged lysosomes within the same neurons (Fig. S1f). We investigated then the expression of p62/SQSTM1, a longlived protein known to be selectively degraded by autophagy.28 Addition of STS induced a progressive and strong reduction in p62/SQSTM1 suggesting an increase in the whole process of autophagy (Fig. 2D). To further confirm that the STS-induced autophagosome increase was due to autophagy induction and not impaired autophagosome-lysosome fusion, we used bafilomycin A1 (BA1), a vacuolar type H+ -ATPase inhibitor that prevents lysosome acidification and autophagosome-lysosomal fusion. Treatment for 12 h with BA1 (300 nM), at which concentration autophagosome-lysosomal fusion is completely blocked, led as expected to a substantial accumulation of autophagosomes (LC3-II level) and of p62/SQSTM1 (Fig. 2E). Importantly, when this 12 h BA1 treatment was combined for the last 6 h with STS, it resulted in a greater increase in the LC3-II level than with BA1 or STS alone and in the prevention of p62 degradation normally induced by STS (Fig. 2E). To evaluate whether the increase in autophagosome formation induced by STS also led to maturation into autolysosomes, we used an RFP-GFP-LC3 DNA construct in order to discriminate between early and late autophagosomes (Fig. 2F). Following STS treatment, besides confirming a global increase in the total number of autophagosomes, STS increased both the number of early autophagosomes (GFP+ RFP+ LC3positive dots) and the number of late autophagosomes (GFP- RFP+ LC3-positive dots) demonstrating that the maturation of autophagosomes into autolysosomes is also increased upon STS treatment. Taken together, these results confirm that STS-induces de novo formation of autophagosomes and enhanced autophagic flux. Activation of lysosomes and caspase-3 in the same neuron. Double immunolabeling for Lamp1 and cleaved caspase-3 revealed that neurons displaying condensed chromatin were always positive for cleaved caspase-3 and that some of them were also characterized by enhanced Lamp1 labeling 12 h after STS treatment (Fig. S1g). Since neurons with noncondensed chromatin also often presented strong Lamp1 staining but were negative for cleaved caspase-3, and since some with condensed chromatin were only very weakly positive for Lamp1, these observations confirm our protein gel blot results (Fig. 1B and Fig. S1b) suggesting that enhanced autophagy precedes caspase-dependent apoptosis and that Lamp1 staining dissipates as apoptosis progresses.

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Figure 1. Time-course of cell death, caspase-3 activation and autophagosome increase in staurosporine-treated cultured cortical neurons. (A) Left part: quantification of pyknotic nuclei as a percentage of all nuclei, following exposure to 1 μM staurosporine (STS), showing a progressive increase in neuronal death (ct: 10 ± 2%, 6 h: 34 ± 3%, 12 h: 84 ± 3%, 24 h: 98 ± 1%; n ≥ 12). Right hand parts: representative images of Hoechst-stained nuclei after 12 h of DMSO (ct) or STS. Arrows indicate pyknotic nuclei. (B) Representative immunoblots for LC3 and cleaved caspase-3 and the corresponding quantification showing that STS treatment significantly increases cleaved caspase-3 (3 h: 365 ± 25%, 6 h: 675 ± 78%, 12 h: 1,014 ± 77%, 24 h: 1,499% ± 143%) and LC3-II (3 h: 260 ± 19%, 6 h: 361 ± 44%, 12 h: 380 ± 22%, 24 h: 249 ± 15%) (n ≥ 6). Values are expressed as a percentage of control. (C) Upper parts: Confocal images of LC3 immunolabeling (green) of two representative cultured cortical neurons at different time points following STS treatment. STS exposure induces a progressive increase in LC3-positive dots (green). Hoechst-stained nuclei are blue. Lower part: quantification of the number of the LC3-positive dots per neuron, demonstrating a strong increase in the number of autophagosomes over the first 12 h of STS treatment (ct: 5.9 ± 1.4%, 3 h: 35 ± 5.3%, 6 h: 40.4 ± 2.6%, 12 h: 47.1 ± 4.5%; 24 h: 17.9 ± 2.2%; n ≥ 5, 20 neurons per experiment). Results are mean ± SEM, **p < 0.01, ***p < 0.001, vs. control, Student’s t-test. Bars = 20 μM.


Mitophagy is not involved in STS-induced neuronal apoptosis. Since, in some conditions, autophagy can inhibit apoptosis by removing dysfunctional mitochondria or aggravate it by an excessive destruction of mitochondria, we tested if STS induced mitophagy. We examined whether the mitochondrial marker mtHSP70 colocalized with autophagosome/lysosome markers. Double-immunofluorescent analysis revealed very few colocalizations of mtHSP70, either with autophagosomes (dots of LC3

staining) or with the lysosomal marker cathepsin B (Fig. S2A and B). Pharmacological inhibition of autophagy and caspases are both protective but more efficient when combined. To further investigate the role of enhanced autophagy in STS-induced neuron death, we investigated the effects of 3-methyladenine (3-MA), a widely used autophagy inhibitor, and of a broad spectrum caspase inhibitor, Q-VD-OPH. Pre-treatment with 3-MA

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Figure 2 (See opposite page). Staurosporine treatment increases lysosomal activity and autophagic flux in cultured cortical neurons. (A) Enzymatic assay for acid phosphatase (AP), performed on protein extracts of cultured neurons (10 μg) following exposure to 1 μM staurosporine (STS), indicating a moderate and transient increase in AP activity at 3 h (125 ± 6%) and 6 h (131 ± 2%). Values are expressed as a percentage of control (ct, 0.1% DMSO for 24 h), **p < 0.01, vs. control; Student’s t-test, n ≥ 4. (B) Cytochemistry for AP 6 h after STS treatment showing an increase in the number of large AP-positive puncta (right parts). Punctum areas have been quantified and classified in three size-groups: small (≤0.3 μm2), intermediate (>0.3 and ≤0.9 μm2) and large (>0.9 μm2). STS treatment has substantially increased the percentage of large puncta (left part). Values represent percentage of AP-positive puncta per size-group per neuron. **p < 0.01 between DMSO and STS; Student’s t-test, n ≥ 15. (C) Cytochemistry for β-N-acetylhexosaminidase showing an increase in the size of β-N-acetylhexosaminidase-positive dots in STS-treated neurons (6 h) compared with control. (D) Representative immunoblots for p62/SQSTM1 (upper part) and the corresponding quantification (lower part) showing that STS treatment significantly decreases p62 expression (3 h: 75 ± 5%, 6 h: 52 ± 2%, 12 h: 30 ± 1%, 24 h: 18% ± 1%) (n ≥ 4). Values are expressed as a percentage of control. (E) Representative immunoblots against LC3 and p62/SQSTM1 and the corresponding quantifications demonstrating that addition of bafilomycin A1 (BA1, 300 nM, 12 h) to the cultures increases the level of LC3-II (BA1: 717 ± 41%) and p62/SQSTM1 (BA1: 169 ± 18%) relative to control (ct) indicating a blockage of autophagic degradation (n ≥ 5). Combination of BA1 and STS treatment (6 h after exposure to BA1) results in a greater increase in LC3-II than treatment with STS or BA1 alone (BA1 + STS: 995 ± 126%; *p < 0.05; Student’s t-test) and mitigates the STS-induced degradation of p62/SQSTM1 (STS: 51 ± 3%, BA1 + STS: 141 ± 18%, **p < 0.01; Student’s t-test) confirming that STS induces functional autophagy. Results are expressed as mean ± SEM. Bars = 20 μm. (F) Upper parts: confocal images of a representative cultured cortical neuron transfected with GFP RFP-LC3 DNA construct at different time points following STS treatment. Lower part: Quantification of the number of LC3-positive dots per neuron following STS treatment. Total LC3-positive dots per neuron (GFP+ RFP+ and GFP- RFP+ LC3-positive dots): ct: 10.3 ± 1.9%, 6 h: 35.4 ± 3.6%, 12 h: 71.3 ± 4%; early autophagosomes (GFP+ RFP+ LC3-positive dots): 6.3 ± 1.4%, 6 h: 24.2 ± 2.8%, 12 h: 47 ± 3.1%; late autophagosomes (GFP- RFP+ LC3-positive dots): 4.5 ± 1.4%, 6 h: 11.2 ± 1.5%, 12 h: 24.3 ± 1.8%. Results are mean ± SEM, n = 4 independent experiments (20 neurons per experiment). ***p < 0.001, vs. control, Student’s t-test. Bars = 10 μM.

(10 mM) resulted in a significant neuronal protection as measured by counts of pyknotic nuclei (Fig. 3A) or by the LDH release assay (Fig. S3a) 12 h after exposure to STS. Pre-treatment with Q-VD-OPH (25 μM) resulted in a better protection than with 3-MA, and combination of both inhibitors was more efficient than either alone since neuronal death was almost totally prevented (Fig. 3A and Fig. S3a). Pre-treatment with Q-VD-OPH completely prevented caspase-3 activation as shown by immunoblot for cleaved caspase-3 (Fig. 3B) and for caspase-dependent α-fodrin cleavage (Fig. S3d), without any effect on the LC3-II level (Fig. S3c) or on calpain activation (Fig. S3d). In addition to decreasing autophagy (Fig. S3b), 3-MA treatment significantly reduced caspase-3 activation [shown by decreases in both cleaved caspase-3 (Fig. 3B) and caspase-3-mediated α-fodrin cleavage (Fig. S3d)], as well as calpain activation [shown by the reduction in calpain-mediated α-fodrin cleavage (Fig. S3d)]. We evaluated also the effect of 3-MA on caspase-independent apoptosis by investigating apoptosis-inducing factor (AIF) translocation to the nucleus after STS treatment. 3-MA strongly reduced the AIF translocation normally induced 6 h after STS treatment (Fig. 4D) whereas Q-VD-OPH has no effect on it (not shown). The strong neuroprotective effect of 3-MA and Q-VD-OPH in combination was reflected in a robust decrease in both apoptosis (caspases and AIF) and calpains (Fig. 3B and Fig. S3d). Knockdown of Atg5 or Atg7 reduces STS-induced neuronal apoptosis and is strongly protective when combined with caspases inhibition. In view of the possible nonspecific effects of 3-MA in addition to the inhibition of autophagy, we performed a knockdown of different autophagy genes with lentiviral vectors encoding short hairpin RNAs (shRNAs): Atg5, Atg7 and Becn1/ Atg6. Atg5 knockdown was confirmed by Q-RT-PCR, which showed that Atg5 mRNA expression in shRNA-Atg5 transduced neurons was inhibited by 55% (Fig. 4A). Downregulation of Atg5 decreased autophagosome formation in response to STS treatment as shown by a decrease in punctate sapphire-LC3 (Fig. 4B) and by a reduction of about 30% in LC3-II level

after STS exposure (Fig. 4C). Atg5 knockdown resulted also in decreased activation of caspase-3, as shown in protein gel blots by reduced immunoreactivity of both cleaved caspase-3 (Fig. 4D) and caspase-specific α-fodrin fragment (Fig. S4a). Downregulation of Atg5 decreased significantly the number of pyknotic nuclei (Fig. 4E) or release of LDH (Fig. S4d) than control-infected neurons 12 h after STS treatment, implying a higher survival rate. Moreover, pre-treatment of cultures with Q-VD-OPH had a strong protective effect when combined with Atg5 knockdown, reducing both the percentage of pyknotic neurons (Fig. 4E) and the release of LDH (Fig. S4d). This synergistic neuroprotective effect indicates that caspaseindependent autophagy-related mechanisms are involved in the execution of STS-induced neuron death. Similar results were obtained with the combination of two Atg7 shRNAs which gave a very strong inhibition of Atg7 expression as shown by Q-RTPCR (Fig. 5A) or by protein gel blot (Fig. 5B). Neurons transduced with Atg7 shRNAs showed not only a strongly decreased STS-induced autophagosome formation as demonstrated by a decrease in sapphire-LC3-positive dots (Fig. 5C) and in LC3-II level (around 58%) by immunoblots (Fig. 5D), but also inhibition of caspase-3 (Fig. 5E and Fig. S4b) and calpain (Fig. S4b). Cell death quantification demonstrated strong reduced toxicity in Atg7 knockdown neurons as shown by a decrease in pyknotic nuclei 12 h after STS (Fig. 5F) and by measurement of LDH-release (Fig. S4e). Moreover, knockdown of Atg5 or Atg7 strongly prevented STS-induced intra-nuclear AIF accumulation as shown in Figure 6. These results suggest that autophagy plays a role upstream of apoptosis. To confirm a death promoting role of excessive autophagy following STS, we investigated whether the upregulation of Atg7 gave opposite results to Atg7 downregulation (Fig. 7). Neurons transduced with Atg7-myc lentiviral vector, which strongly overexpressed Atg7 (Fig. 7A), displayed enhanced autophagosome formation as shown by an increase in LC3-positive puncta following immunolabeling (Fig. 7B) or by LC3-II level in protein gel blot analysis (Fig. 7C). Moreover, this effect was enhanced in the presence of STS and Atg7 upregulation sensitized the



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© 2011 Landes Bioscience. Do not distribute. Figure 3. Combination of 3-methyladenine and Q-VD-OPH is highly protective against staurosporine neurotoxicity. (A) Quantification of pyknotic nuclei 12 h after staurosporine (STS) exposure showing that pre-treatment with 3-methyladenine (3-MA, 10 mM) or Q-VD-OPH (25 μM) reduces STSinduced neuronal death and combination of the two inhibitors affords greater neuroprotection than either treatment alone. Counts of pyknotic nuclei are expressed as a percentage of the total number of nuclei (ct, 0.1% DMSO) (n ≥ 7). (STS: 82 ± 2%; STS + 3-MA: 58 ± 2%; STS + Q-VD-OPH: 47 ± 3%; ST S + 3-MA + Q-VD-OPH: 28 ± 2%). (B) Representative protein gel blot and the corresponding quantification of cleaved caspase-3 12 h after STS exposure, demonstrating that not only Q-VD-OPH but also 3-MA pre-treatments significantly decrease caspase-3 activation (STS: 1,091 ± 160%; STS + 3-MA: 717 ± 92%; STS + Q-VD-OPH: 108 ± 31%; STS + 3-MA + Q-VD-OPH: 98 ± 17%). Values are expressed as a percentage of control (0.1% DMSO). Results are mean ± SEM, *p < 0.05; **p < 0.01, vs. STS-treated, Student’s t-test, n = 3. (C) Quantification of AIF nuclear translocation following 6 h of STS (right part) (ct: 15 ± 1%; STS: 48 ± 2%, STS + 3-MA: 19 ± 1%) and representative images of neurons immunostained for AIF (green) and Hoechst (blue) showing that pretreatment with 3-MA strongly reduces AIF nuclear translocation. Results are mean ± SEM, ***p < 0.001, vs. control, Student’s t-test. n ≥ 20. Bar = 20 μm.

neurons to STS-induced apoptosis since both the level of cleaved caspase-3 (Fig. 7C) and the number of pyknotic nuclei (Fig. 7E) were markedly increased; even in the absence of STS there was a moderate induction of apoptosis when Atg7 was overexpressed (Fig. 7C and E). The sensitization to STS induced by Atg7 overexpression could be prevented by the addition of 3-MA or by Atg5 knockdown (Fig. S6). Taken together, the data from the Atg5 and Atg7 knockdown experiments are similar to those obtained with the pharmacological inhibition of autophagy, and demonstrate an involvement of autophagy in the mediation of STS-induced neuronal apoptosis through both caspase-dependent and independent pathways.

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Autophagy enhanced in STS-induced neuronal apoptosis is Beclin 1-independent but Beclin 1 knockdown sensitizes neurons to caspase-dependent apoptosis. It has been shown that Beclin 1 can be involved in autophagic cell death following some stimuli.12,29 Surprisingly, strong downregulation of Beclin 1 (by 75%) (Fig. 8A) did not affect STS-induced autophagosome formation (Fig. 8B and C), whereas it strongly inhibited autophagosome formation induced by rapamycin (Fig. 8D) as expected30 or in basal conditions (Fig. S7c). These results indicate that while Beclin 1 is involved in the regulation of the mammalian target of rapamycin (m-TOR) dependent autophagic pathway in neurons, STS-induced autophagy is Beclin 1-independent.

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© 2011 Landes Bioscience. Do not distribute. Figure 4. Knockdown of Atg5 reduces staurosporine-induced autophagy and apoptosis. (A) Q-RT-PCR assay for Atg5 in primary cortical neurons transduced with a lentiviral vector encoding shRNA targeted to Atg5 showing that Atg5 shRNA decreases Atg5 mRNA expression (47 ± 0.6%) (***p < 0.001, vs. infection control, Student’s t-test, n = 5). (B) Confocal images of staurosporine (STS)-treated cortical neurons expressing sapphire-LC3 and immunolabeled with an antibody against GFP, showing that downregulation of Atg5 decreases strongly the presence of sapphire-LC3-positive dots. Bar = 10 μm. Expression of (C) LC3-II (empty vector + STS: 308 ± 38%; shRNA Atg5 + STS: 214 ± 9%) and (D) cleaved caspase-3 (empty vector + STS: 834 ± 42%; shRNA Atg5 + STS: 444 ± 49%) are both significantly reduced in shRNA Atg5-treated neurons 12 h after the application of STS as shown by representative protein gel blots and their corresponding quantifications. Values are expressed as a percentage of control (0.1% DMSO). Results are mean ± SEM, **p < 0.01, shRNA Atg5 STS vs. infection control STS, Student’s t-test, n ≥ 7. (E) Quantification of pyknotic nuclei 12 h after STS exposure showing significantly reduced cell death in STS-treated neurons with Atg5 knockdown and near complete protection together with Q-VD-OPH (empty vector + STS: 88 ± 1%; shRNA Atg5 + STS: 59 ± 2%; empty vector + STS + Q-VD-OPH: 60 ± 2%; shRNA Atg5 + STS + Q-VD-OPH: 30 ± 1%). Results are mean ± SEM, ***p < 0.001, Student’s t-test, n ≥ 6.

We observed that Beclin 1 knockdown was accompanied by a strong activation of caspase-3 (compare with the control infected neurons) in the presence and even in the absence of STS (Fig. 8E and Fig. S4c), thus sensitizing neurons to death (increase in pyknotic nuclei and LDH-release (Fig. 8F and Fig. S4f). Q-VD-OPH treatment very efficiently reduced STS-induced toxicity in neurons transduced with Beclin 1 shRNA (Fig. 8F).

Downregulation of Beclin 1 in STS-treated neurons also caused a 2.6-fold stronger activation of calpain compared with controlinfected neurons, as shown by an increase in calpain-dependent cleavage of α-fodrin (Fig. S4c). Overexpression of Beclin 1 did not result in any significant effects on the STS-induced apoptosis and autophagy (neurotoxicity, LC3-II accumulation, caspase and calpain activation)


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© 2011 Landes Bioscience. Do not distribute. Figure 5. Knockdown of Atg7 inhibits staurosporine-induced autophagy and apoptosis. (A) Q-RT-PCR assay for Atg7 in primary cortical neurons transduced with lentiviral vectors encoding shRNAs targeted to Atg7 showing that shRNAs against Atg7 strongly decreases Atg7 mRNA relative to control infection (13 ± 0.5%) (n = 5). (B) Representative protein gel blot and corresponding quantification showing the downregulation of Atg7 in neurons transduced with lentiviral shRNAs against Atg7 (47 ± 2%) compared with control neurons (n = 9). (C) Confocal images of STS-treated cortical neurons expressing sapphire-LC3 and immunolabeled with an antibody against GFP demonstrating that downregulation of Atg7 decreases drastically the presence of sapphire-LC3-positive dots. Bar = 10 μm. Expression of (D) LC3-II (empty vector: 293 ± 22%; shRNAs Atg7: 124 ± 12%) and (E) cleaved caspase-3 (empty vector: 565 ± 27%; shRNAs Atg7: 162 ± 6%) are both significantly reduced in neurons treated with Atg7 shRNAs 12 h after the application of STS (1 μM) as shown by representative protein gel blots and their quantifications (n ≥ 6). Values are expressed as a percentage of control (0.1% DMSO). Results are mean ± SEM, ***p < 0.001, Atg7 shRNAs vs. infection control, Student’s t-test. (F) Quantification of pyknotic nuclei 12 h after STS exposure showing significant protection against STS with Atg7 knockdown and almost complete protection when Atg7 knockdown is combined with Q-VD-OPH (empty vector + STS: 88 ± 1%; shRNAs Atg7 + STS: 48 ± 2%; empty vector + STS + Q-VD-OPH: 58 ± 3%; shRNAs Atg7 + STS + Q-VD-OPH: 29 ± 2%). Results are mean ± SEM, ***p < 0.001, Student’s t-test, n ≥ 6.

(Fig. S5) confirming that Beclin 1 is not involved in STS-induced neuronal death. Similar role of Beclin 1-independent autophagy in apoptosis caused by other stimuli. To evaluate whether our results can be generalized beyond apoptosis induced by STS, we investigated the role of autophagy in neuronal death due to two other frequently used apoptotic stimuli, MK801 (40 μM) and etoposide (10 μM).31-33 Both stimuli led not only to an increase in cleaved

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caspase-3, but also in LC3-II level (Fig. 9A and B) and LC3positive puncta (Fig. S8a and b) suggesting an induction of autophagy, which was independent of Beclin 1 (Fig. S8c and d) but Atg7-dependent. Atg7 downregulation prevented the increases in both LC3-II and cleaved caspase-3 following 48 h of treatment with MK801 (Fig. 9C) or etoposide (Fig. 9D) and provided neuroprotection as shown by a reduction in the numbers of pyknotic nuclei (Fig. 9E and F). In contrast, Beclin 1 downregulation

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© 2011 Landes Bioscience. Do not distribute. Figure 6. Knockdown of Atg5 or Atg7 reduces nuclear accumulation of apoptosis inducing factor. (A) Quantification of nuclear apoptosis inducing factor (AIF) staining (average of nuclear fluorescence intensity/μm2) reveals that AIF nuclear translocation stimulated by staurosporine (STS) is strongly reduced in neurons transduced with shRNAs against Atg5 or Atg7 compared with empty vector control (empty vector + STS: 55 ± 3; shRNA Atg5 + STS: 31 ± 2; shRNAs Atg7 + STS cortical: 26 ± 3). Data are mean ± SEM ***p < 0.001, Student’s t-test, n ≥ 10. (B) Representative images of primary cortical neurons stained with anti-AIF antibody (green) and Hoechst (blue) infected with empty vector (left), shRNA Atg5 (middle) and shRNAs Atg7 (right) after 6 h of STS (1 μM) treatment showing a reduction of AIF nuclear translocation. Bars = 20 μm.

increased the activation of caspase-3 following treatment with MK801 or etoposide and increased the numbers of pyknotic nuclei (Fig. S8c and d). The latter results indicate that the autophagic control of apoptosis occurs with other apoptotic stimulations than STS, and may be considered an important contributor to the neuronal apoptotic response. Discussion The present study shows that, following apoptotic stimulation in cortical neurons, autophagy is strongly enhanced and involved in mediating both the caspase-dependent and -independent

components of the apoptosis. Moreover, this induced autophagy is dependent on autophagy genes Atg5 and Atg7 but is Beclin 1-independent, suggesting that a selective and noncanonical pathway of autophagy contributes to neuronal apoptosis. Staurosporine induces autophagy prior to caspase activation. Our observations on caspase-3 and AIF release and protection by caspase-inhibition agree with previous studies on STS-treated on cortical neurons.25,26 Only a few studies have reported STSinduced autophagy in unmodified cells34,35 especially in cortical neurons,36 but in specific conditions, such as in fibroblast mutants incompetent for apoptosis, STS has been reported to cause autophagic cell death that is blocked by 3-MA or by knockdown of Atg5 or Beclin 1.11 Also, in cell lines exposed to a


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© 2011 Landes Bioscience. Do not distribute. Figure 7. Overexpression of Atg7 sensitizes neurons to staurosporine-induced apoptosis. (A) Representative protein gel blot and the corresponding quantification showing the upregulation of Atg7 expression in Atg7-myc-transduced neurons (n = 7). (B) Confocal images of staurosporine (STS)-treated cortical neurons immunolabeled for LC3 showing an increase of LC3-positive dots in Atg7-myc-transduced neurons. Bar = 10 μm. (C) Representative protein gel blots and the corresponding quantifications demonstrating that overexpression of Atg7 increases the expression of both LC3-II (empty vector + STS: 248 ± 12%, Atg7-myc + STS: 362 ± 44%) and cleaved caspase-3 (empty vector + STS: 580 ± 42%, Atg7-myc + STS: 922 ± 77%) (n ≥ 4). (D) Representative protein gel blots and the corresponding quantifications showing the effect of Atg7 overexpression on LC3-II levels with the addition of bafilomycin A1 (BA1) (empty vector + STS + BA1: 604 ± 26%, Atg7-myc + STS + BA1: 752 ± 26%) (n ≥ 4). (E) Quantification of pyknotic nuclei 6 h after STS exposure showing that Atg7 upregulation significantly increased cell death in normal and in STS-treated conditions (empty vector: 21 ± 1%; empty vector + STS: 40 ± 2%; Atg7-myc: 33 ± 2%; Atg7-myc + STS: 69 ± 2%) (n ≥ 26). Results are mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, Student’s t-test.

caspase inhibitor, STS promoted a rapid activation of autophagy prior to apoptosis.37 In the present study we showed not only an early increase in autophagosome formation following STS exposure of cortical neurons but also an activation of the lysosomal pathway, thereby demonstrating an increase in autophagic flux. Moreover, autophagy, lysosomal activation (acid phosphatase, β-hexasominidase, cathepsin D) and apoptosis occurred in the same neurons, although enhanced autophagic and lysosomal features appeared earlier than caspase-3 activation, suggesting that autophagy might be involved in triggering apoptosis. Induced autophagy contributes to neuronal apoptosis. To test this possibility we needed to inhibit autophagy. The only available pharmacological inhibitors of autophagy are PtdIns3K inhibitors, of which the most frequently used for studies of autophagy is 3-MA, although wortmannin and LY294002 are also

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used.6,7,11 These inhibitors reduce autophagy by virtue of their action on class III PtdIns3K, but they also affect the other classes of PtdIns3K including class I, which activates the powerfully anti-apoptotic and anti-autophagic Akt pathway. Therefore, even if these pharmacological inhibitors were rigorously specific for PtdIns3Ks, which is not the case,38 they would still not be specific inhibitors of autophagy. In the present study we used 3-MA, which is in many cases a very effective inhibitor of autophagy, but not always because, by virtue of its inhibition of class I PtdIns3K, it can even promote autophagy, especially after long exposure.39 This may help to explain why the autophagy inhibition by 3-MA was only partial in the present experiments, but the Beclin 1-independence of the autophagy may also be relevant, because class III PtdIns3K interacts with Beclin 1 and the role of class III PtdIns3K in Beclin 1-independent autophagy is

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Figure 8. Knockdown of Beclin 1 does not affect staurosporine-induced autophagy but sensitizes neurons to apoptosis. (A) Representative protein gel blot and the corresponding quantification showing a downregulation of about 73% of Beclin 1 expression in neurons transduced with Becn1 shRNA compared with control neurons. (n ≥ 5). (B) Confocal images of staurosporine (STS)-treated cortical neurons expressing sapphire-LC3 and immunolabeled with an antibody against GFP demonstrating that downregulation of Beclin 1 has no effect on the formation of sapphire-LC3-positive dots. Bar = 10 μm. (C) Expression of LC3-II (empty vector + STS: 301 ± 13%; shRNA Becn1 + STS: 322 ± 19%; n ≥ 5) is not affected by downregulation of Beclin 1, 12 h after STS exposure (1 μM) as shown by representative protein gel blot and its quantifications but (D) is strongly decreased (empty vector: 368 ± 66%; shRNA Becn1: 169 ± 9%, n = 6) following rapamycin treatment (10 μg/ml, 24 h). (E) Expression of cleaved-caspase-3 (empty vector: 84 ± 7%; empty vector + STS: 779 ± 22%; shRNA Becn1: 342 ± 33; shRNA Becn1 + STS: 2,192 ± 143%) is dramatically increased by downregulation of Beclin 1. Beclin 1-enhanced caspase-3 activation following STS treatment is almost completely prevented by Q-VD-OPH (391 ± 94%) (n ≥ 5). Values are expressed as a percentage of control (0.1% DMSO, uninfected) (mean ± SEM), ***p < 0.001, Student’s t-test. (F) Quantification of pyknotic nuclei 12 h after STS exposure showing that Beclin 1 knockdown significantly increases cell death in normal and in STS-treated conditions (empty vector: 23 ± 1%; empty vector + STS: 79 ± 2%; shRNA Becn1: 56 ± 2%; shRNA Becn1 + STS: 100 ± 0.4%) (n ≥ 26). Results are mean ± SEM, ***p < 0.001, Student’s t-test.


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Figure 9. Knockdown of Atg7 inhibits apoptosis induced by etoposide or MK801. Representative protein gel blots and the corresponding quantifications showing that the levels of cleaved caspase-3 and LC3-II are both increased by treatment with (A) MK801 (40 μM) (cleaved caspase-3, 24 h: 611 ± 18%, 48 h: 874 ± 61%; LC3-II, 24 h: 125 ± 4%, 48 h: 175 ± 9%) or with (B) etoposide (10 μM) (cleaved caspase-3, 24 h: 1,096 ± 142%, 48 h: 1,025 ± 140%; LC3-II, 24 h: 124 ± 16%, 48 h: 143 ± 2%) (n ≥ 4). Representative protein gel blots and the corresponding quantifications demonstrating that downregulation of Atg7 prevents the caspase-3 activation and LC3-II increase induced by 48 h of (C) MK801 (cleaved caspase-3, empty vector: 939 ± 38%, shRNA Atg7: 126 ± 10%, LC3-II: empty vector: 281 ± 19%, shRNA Atg7: 80 ± 5%) or of (D) etoposide (cleaved caspase-3, empty vector: 1,422 ± 90%, shRNA Atg7: 202 ± 16%; LC3-II, empty vector: 156 ± 11%, shRNA Atg7: 49 ± 18%) (n = 4). Quantification of pyknotic nuclei 48 h following (E) MK801 (empty vector: 44 ± 1%, shRNA Atg7 21 ± 1%) and (F) etoposide (empty vector: 57 ± 1%, shRNA Atg7 34 ± 2%) treatments shows that Atg7 downregulation provides strong neuroprotection (n ≥ 7). Results are mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, Student’s t-test. 1126

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currently unclear. In view of these limitations of 3-MA as an autophagy inhibitor, it is important that our shRNA-mediated downregulation of Atg5 and Atg7 gave similar results to pharmacological inhibition with 3-MA. All three ways of inhibiting autophagy decreased neuronal apoptotic features and death, and they all strongly enhanced the protection in neurons exposed to Q-VD-OPH. It is now clear that autophagy and apoptosis share several common effectors such as PUMA/Bax,40 FLIP proteins41 or Atg4D.42 Enhanced autophagy can prevent43 or activate8,19,44 apoptosis depending on the conditions. Two studies showed that Atg5 could promote apoptosis induction independently of its role in autophagy. Atg5 interacted with Fas-associated protein with death domain and stimulated caspase-dependent cell death in HeLa cells treated with IFNγ.45 Moreover, in response to several apoptotic stimuli Atg5 has been shown to be cleaved by calpain, after which a cleavage fragment translocates from the cytosol to mitochondria triggering cytochrome c release and caspase activation.21 In the present study, however, calpain-mediated effects seem not to be the cause of caspase activation because treatment with the calpain inhibitor ALLN increased caspase-3 activation rather than reducing it (data not shown). We cannot strictly rule out contributions from other autophagy-independent mechanisms, but the similar reductions of apoptotic features obtained with three different means of autophagy inhibition, 3-MA treatment and Atg7 knockdown as well as Atg5 knockdown, suggest that the autophagic pathway was involved. Furthermore, we have also shown that overexpression of Atg7 leads to increased autophagy and apoptosis even in the absence of an apoptotic agent. Moreover, blockade of autophagy by Atg5 knockdown prevents this sensitization induced by Atg7 overexpression, demonstrating that the pro-death effects of Atg7 overexpression are mainly autophagy-dependent. Although most of our experiments involved STS-induced autophagy, we tested other apoptotic stimulations to confirm the generality of the phenomena. Using MK801 and etoposide we again demonstrated the efficacy of Atg7 knockdown for reducing caspase-3 activation and nuclei pyknosis. Thus, our conclusions are relevant to other apoptotic stimulations than STS, suggesting that autophagy, or autophagic signaling, is an important inducer of neuronal apoptosis in multiple situations. The autophagy-apoptosis link does not depend on Beclin 1. Another important link between apoptosis and autophagy is the interaction between Bcl2 and Beclin 1, because Bcl2 is both anti-apoptotic and anti-autophagic. Beclin 1 was first identified as a protein binding to Bcl2 through its BH3 domain. In basal conditions Beclin 1 is sequestered by Bcl2 and under stress conditions Beclin 1 is released and induces autophagy.46 However, although Bcl2 has a strong anti-autophagic effect, overexpression of Beclin 1 did not affect apoptosis47 as observed also in the present study. Thus, although Beclin 1 might theoretically be pro-apoptotic by sequestering Bcl2 and thereby counteracting its anti-apoptotic action, it is apparently not sufficiently abundant to actually do so in the present experiments. Furthermore, our results concerning Beclin 1 downregulation are the very opposite of what might be predicted from its sequestering of Bcl2, because

the downregulation strongly induced neuronal apoptosis in basal conditions as well as sensitizing neurons to STS-induced apoptosis. Our observations thus suggest an essential role of Beclin 1 for neuronal survival in basal and STS conditions, and similar results with Beclin 1 downregulation have been suggested previously in different models.43,48 Some recent observations on the death of differentiated neural precursor cells resemble ours to the extent that the cell death was reduced by the knockdown of Atg7 but not Beclin 1.49 However, the experimental model was very different from ours, involving treatment with chloroquine or bafilomycin A1, both of which reduce autophagic flux, either by inhibiting lysosomal functions (chloroquine) or by blocking the fusion of autophagosomes to lysosomes (bafilomycin A1). This contrasts with our experiments in which STS induced an increase in autophagic flux in cortical neurons including an enhanced lysosomal degradative activity, so the relationship between the two experimental situations is unclear. Induced-autophagy in neuronal apoptosis is Beclin 1-independent. Beclin 1 is an important autophagy regulator playing several different roles along the autophagic process. Beclin 1 can act in the induction step prior to autophagy,49,50 in autophagosome formation51 and in autophagosome maturation.50 Our experiments with Beclin 1 knockdown showed that, although a ~75% reduction in Beclin 1 protein efficiently reduced rapamycin-induced autophagy, it did not affect autophagosome formation after exposure to STS, indicating that STS triggers a Beclin 1-independent autophagy in dying cortical neurons. We have similar evidence for Beclin 1-independent autophagy following MK801 and etoposide treatments, supporting the generality of the phenomenon. Beclin 1-independent autophagy has been reported in only a few previous cases. In SH-SY5Y cells exposure to the neurotoxin 1-methyl-4-phenylpyridinium (MPP+) provoked autophagy and cell death that were insensitive to PtdIns3K inhibition or Beclin 1 knockdown.52,53 Likewise, autophagy that is insensitive to knockdown of Beclin 1 or of its binding partner hVps34 (i.e., class III PtdIns3K) is involved in the caspaseindependent death of cancer cells induced by resveratrol,54 in the hydrogen peroxide induced death of GSH-depleted RAW 264.7 cells,55 in the obatoclax-induced death of various cancer cells56 and in HeLa cells treated with an inhibitor of the the BH3binding groove of Bcl-X L/Bcl-2.57 Increased expression of Beclin 1 has sometimes been considered a marker of enhanced autophagy in neurons in various neurodegenerative conditions including closed head injury, traumatic brain injury, spinal cord injury and focal cerebral ischemia.58-60 The existence of Beclin 1-independent autophagic mechanisms emphasizes the limitations of Beclin 1 as an autophagy marker. The reason for the lack of effect of Beclin 1 knockdown on autophagy and neuronal death is unknown, but a contributing factor may be the disactivation of Beclin 1 by caspase-mediated cleavage, which occurs in cell lines exposed to various apoptotic stimuli including STS.61 We do in fact have evidence that Beclin 1 was cleaved in our neurons exposed to STS for 12 h and that this cleavage was caspase-dependent (Fig. S9). The amount of full-length Beclin 1 was only moderately decreased, however, so



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Beclin 1 depletion would only partially explain the lack of effect of Beclin 1 knockdown, and would not at all explain their sensitization to STS-induced apoptosis. In conclusion, our study highlights the importance of checking for autophagy involvement when apoptosis is induced. It shows, with three different apoptotic stimuli, that Beclin 1-independent Atg5/Atg7-dependent autophagy (or at least autophagic signaling) is an important inducer of both the caspase-dependent and -independent components of neuronal apoptosis. The longterm prospect of being able to inhibit both components by inhibiting only one selective pathway of autophagy opens new avenues for clinically useful neuroprotection.


Materials and Methods Lentiviral vectors and virus production. CDNAs encoding full-length rat Atg7 (GenBankTM NM_001012097) and Becn1 (GenBankTM NM_053739) with C-terminal Myc-tag was created using PCR with specific primers (Atg7: nucleotides 16–35 (forward), 2,092–2,112 (reverse); Becn1: nucleotides 225–243 (forward), 1,549–1,570 (reverse)) to which the myc-tag encoding sequence was added. Resultant cDNAs and rat MAP Lc3B (GenBankTM NM_022867) cDNA fused to the C-terminus of sapphire fluorescent protein cDNA (a generous gift of Dr. Shibata and Prof. Uchiyama) were cloned into a self-inactivating lentiviral transfer vector under the control of mouse phosphoglycerate kinase 1 (Pgk1) promoter (SIN-W-PGK),61 using the pENTR/DTOPO cloning Kit (Invitrogen, K240020) and the LR recombination reaction (Gateway System, Invitrogen, 11791020).62 shRNAs specific for rat genes from TRC (the RNAi consortium) library in pLKO lentiviral vectors were used as follows: TRCN0000099431 for Atg5 (GenBankTM NM_001014250.1), TRCN0000033552 for Becn1 (GenBankTM NM_053739.2), and a combination of TRCN0000092163 and TRCN0000092166 for Atg7 (GenBankTM NM_001012097) (Openbiosystems). Selfinactivating lentiviral vectors were produced as described previously in reference 63. Primary cortical neuronal cultures. All experiments were performed in accordance with the Swiss Law for the protection of animals and were approved by the Vaud Cantonal Veterinary Office. Primary neuronal cultures were prepared from the cortices of 2-d-old Sprague-Dawley rats and maintained as described previously in reference 64. For biochemical analyses and LDH release measurement, neurons were plated at a density of 6 x 105 cells/dish (35 mm poly-d-lysine precoated dishes, BD Biosciences, 356467). For immunocytochemistry, neurons were plated at a density of 1.5 x 104 cells/well of 24 well plates (or 9.6 x 104 cells/well for lentiviral infection experiments) on 12 mm coverslips coated with 0.01% poly-l-lysine (Sigma, P4832). Cells were maintained at 37°C with a 5% CO2-containing atmosphere for 12 d, and half of the media was changed every 3–4 d. For lentiviral-mediated protein expression and knockdown, cultures were infected on day in vitro (DIV) 7 using 50 ng of p24/ml culture medium for each vector, and half the medium was replaced on DIV 11. Empty vectors were used as an infection control. Control infection did not cause any strong effect on

LDH release, or on the expression of autophagy marker LC3-II, or cell death markers (cleaved caspase-3 or calpain or caspase-3 specific α-fodrin cleavage fragments) compared with noninfected neurons after staurosporine treatment (Table S1). Pharmacological treatment. At DIV 11–12 primary cortical neurons were exposed to 1 μM staurosporine (STS) (SigmaAldrich, S4400) or 0.1% DMSO (Sigma-Aldrich, 154938) in complete culture medium for indicated times. Where specified, cells were pre-treated for 1 h prior to STS application with 25 μM of the pan-caspase inhibitor Q-VD-OPH (Quinoline-ValAsp(ome)-Ch2-O-phenoxy; MP Biomedicals, 030PH10903) or 10 mM of the autophagy inhibitor 3-methyladenine (3-MA) (Sigma-Aldrich, M9281). Bafilomycin A1 (Enzo Life Sciences, ALX-380-030-M001) was applied for 6 h prior to STS. For the other treatments, primary cortical neurons were incubated with 10 μg/ml rapamycin (Sigma, R0395) for 24 h, with 40 μM MK801 (Sigma-Aldrich, M107) or 10 μM etoposide (SigmaAldrich, E1383) for 24 h and 48 h. Immunoblotting. Protein samples were harvested in lysis buffer [20 mmol/L HEPES, pH 7.4, 10 mmol/L NaCl, 3 mmol/L MgCl2, 2.5 mmol/L EGTA, 0.1 mmol/L dithiothreitol, 50 mmol/L NaF, 1 mmol/L Na 3VO4, 1% Triton X-100 (reagents from Sigma-Aldrich), and a protease inhibitor cocktail (Roche, 11873580001)]. Lysates were sonicated and protein concentration was determined using a Bradford assay. Proteins (20–40 μg) were separated by SDS-PAGE on a 10% or 14% polyacrylamide gel, and analyzed by immunoblotting. The following primary antibodies were used for protein immunodetection: anti-Atg7 (sc-33211, 1/1,000) rabbit polyclonal, anti-Beclin 1 (sc-11427, 1/1,000) and anti-α-tubulin (sc-8035, 1/5,000) mouse monoclonal antibodies from Santa Cruz Biotechnology; anti-LC3 (ab48394, 1/3,000) rabbit polyclonal antibody from Abcam; antiactive caspase-3 (#9661, 1/1,000) and anti-AIF (#4642, 1/1,000) rabbit polyclonal antibodies from Cell Signaling Technology; anti p62/SQSTM1 (P0067, 1/1,000) rabbit polyclonal antibody from Sigma-Aldrich and anti-fodrin (#FG6090, 1/3,000) mouse monoclonal antibody from Biomol (Enzo Life Sciences). After incubation with primary antibody, the following secondary antibodies were applied: polyclonal goat anti-mouse or goat antirabbit IgG conjugated with IRDye 680 (LI-COR, B70920-02) or IRDye 800 (LI-COR, 926-32210). Protein bands were visualized using the Odyssey Infrared Imaging System (LI-COR). Odyssey v1.2 software (LI-COR) was used for densitometric analysis. OD values were normalized with respect to tubulin and expressed as a percentage of values obtained for noninfected neurons treated with 0.1% DMSO (100%). All data were expressed as mean ± SEM. Data were analyzed statistically by one-way ANOVA followed by Student’s t-test (2-tailed, 2-sample and unequal variance). Immunocytochemistry. Primary cortical neurons cultured on glass coverslips were fixed with 4% paraformaldehyde in PBS (pH 7.4) for 15 min on ice, and incubated for blocking and permeabilization in PBS with 10% donkey serum and 0.1% Triton X-100 for 1 h. Cells were incubated with primary antibodies diluted in 5% of donkey serum, 0.1% Triton X-100 in PBS overnight at 4°C. The following primary antibodies were used:


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anti-mtHSP70 (ma3-028, 1/100) mouse monoclonal antibody from ABR (Affinity Bioreagents); anti-cathepsin B (#06-480, 1/200) and anti-cathepsin D (#06-467, 1/200) rabbit polyclonal antibody from Upstate Biotechnology; anti-NeuN (#MAB377, 1/500) mouse monoclonal antibody from Chemicon; anti-GFP (#AB3080, 1/500) rabbit polyclonal antibody and anti-AIF rabbit polyclonal antibody (#AB16501; 1/200) from Millipore (Billerica, MA USA). Anti-LC3 was a generous gift from Prof. Yasuo Uchiyama (Tokyo, Japan). Alexa Fluor 488 donkey-antirabbit (Invitrogen, A21206) and Alexa Fluor 594 donkey-antimouse (Invitrogen, A21203) secondary antibodies, diluted in PBS with 1% of donkey serum (1/1,000), were applied for 2 h at room temperature. Coverslips were mounted with FluoroSave (Calbiochem, 345-789-20) and analyzed with either Leica SP5 AOBS or Zeiss LSM 510 Meta confocal laser-scanning microscopes. Images were then processed by with Adobe Photoshop 5.0. Plasmid transfection. Cells on 12 mm coverslips were transfected using Lipofectamine 2000 (Invitrogen, 11668-019) as described previously in reference 65. Time-lapse microscopy. Cortical neurons were plated onto poly-l-lysine-coated glass coverslips that had previously been attached with hot Vaseline/wax (50:50) to 35 mm plastic culture dishes in which holes 15 mm in diameter had been machine drilled.64 On DIV 12, dishes were placed on the stage of a Nikon TE300 microscope. Temperature was maintained at 37°C and CO2 at 5% using a microscope incubator system (Solent Scientific Limited). Selected neurons were imaged with a 40x objective at 5 min intervals. Images were captured with an Orca-ER cooled CCD camera (Hamamatsu). All peripherals were controlled with Metamorph software (Universal Imaging). Images were processed with either Adobe Photoshop or Metamorph. Cytochemistry for lysosomal enzymes. DIV 12 cortical neurons plated on coverslips were washed and fixed at different times after STS treatment in 2% glutaraldehyde and 1% paraformaldehyde diluted in cacodylate buffer (0.1 M, pH 7.4), washed three times in 0.1 M cacodylate buffer (pH 7.3) and stained as previously described in reference 2. For acid phosphatase histochemistry coverslips were incubated for 2 h at 37°C in 0.01 M Na-β-glycerophosphate and 8 μM lead nitrate in sodium acetate buffer (50 mM, pH 5), rinsed in distilled water and immersed in ammonium sulphide (0.5%) for 30 s, rinsed again and mounted with FluoroSave. Controls were performed using sodium fluoride (1 M) to verify the labeling specificity. No staining was detected in any of these controls. For β-N-acetylhexosaminidase cytochemistry coverslips were incubated for 1 h at 37°C in Fast Red Violet salt solution [0.63 M ethylene glycol monomethyl ether, 1% polyvinyl pyrrolidone, 0.1 M citric acid-citrate buffer pH 4.4, 1.6 M sodium chloride, 2.7 mM Fast Red Violet LB salt (Sigma, F3381)] using 0.5 mM Naphthol-AS-BI-N-acetyl-β-D-glucosaminide (Sigma, N4006) as the substrate, post-fixed for 10 min. in 10% formaldehyde, dehydrated in graded alcohols and mounted in Eukitt medium. Controls without the substrate or at basic pH gave no staining. Image acquisition was performed with an Axiovision Zeiss microscope. Quantification of lysosomal enzyme staining was

performed using Image J software and images were then processed with Adobe Photoshop 5.0. Enzyme assay for acid phosphatase. Protein samples were prepared in the same lysis buffer as for immunoblotting but without NaF and Na3VO4, as previously described in reference 23. 10 μg of proteins diluted in sodium acetate buffer (0.1 M, pH = 5.5) with 20 mM PNPP (p-Nitrophenyl Phosphate) was incubated for 2 h at 37°C in a final volume of 400 μl. The reaction was stopped by the addition of 50 μl 2 mol/L NaOH and absorbance was measured at 405 nm. Quantitative real-time PCR analysis. RNA was isolated by RNAeasy kit (Qiagen, 74106) using the manufacturer’s instructions. For cDNA synthesis the High capacity cDNA Reverse Transcription kit was used (Applied Biosystems). Samples were analyzed using Tagman expression assays for β-actin, Rn00667869_m1; Atg5, Rn01767063_m1; Atg7, Rn01492725_ m1. Gene expression was measured from 5 biological repeats and technical triplicates amplified on a 7900HT Real-Time PCR System with SDS 2.3 software (Applied Biosystems). Relative expression values (V) were calculated by the ΔCt method and normalyzed to β-actin expression as previously described in reference 66. Quantification of cell death. Cytotoxicity was calculated by measurements of lactate dehydrogenase (LDH) release in the medium by using the CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega, G1780) as previously described in reference 64. Data are expressed as a percentage of the LDH value of noninfected neurons treated with 0.1% of DMSO. Neuronal death was also evaluated by counting DAPI-stained pyknotic nuclei. Cells were examined using a Zeiss Axioplan 2 imaging microscope. Total number and pyknotic-positive nuclei were then counted using ImageJ software. Results were expressed as a percentage of pyknotic nuclei (relative to the total number of nuclei). Data were expressed as mean ± SEM and analyzed statistically by one-way ANOVA followed by Student’s t-test (2-tailed, 2-sample and unequal variance). Quantification of AIF nuclear translocation. AIF nuclear staining was examined using a Zeiss Axioplan 2 imaging microscope. Measures of AIF nuclear fluorescence were performed using ImageJ software and a 60x magnification objective and then expressed as an average of fluorescence intensity/μm2. All data were expressed as mean ± SEM and analyzed statistically by one-way ANOVA followed by Student’s t-test (2-tailed, 2-sample and unequal variance). Quantification of LC3, Cathepsin B and mtHSP70 labeling. Confocal images of immunocytochemistry against LC3, cathepsinB and mtHSP70 or of transfected neurons with mRFPGFP-LC3 DNA construct on coverslips of cortical neuronal cultures at different time points after STS treatment (0, 3, 6, 12 and 24 h) were acquired using a Zeiss LSM 710 Meta confocal laserscanning microscope. LC3, cathepsinB and mtHSP70-positive dots were then analyzed using ImageJ software and expressed as a number of positive dots per neuron. All data were expressed as mean ± SEM and analyzed statistically by one-way ANOVA followed by Student’s t-test (2-tailed, 2-sample and unequal variance).



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Statistical analysis. Data represent the mean values ± SEM. Data were analyzed statistically by one-way ANOVA followed by Students’ t-test (2-tailed, 2-sample and unequal variance) when ANOVA showed significant differences between the groups (p < 0.05). p < 0.05 was chosen as the threshold for statistical significance. In the case of multiple comparisons, Bonferroni’s correction was applied. Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgements

We thank Coralie Rummel, Florine Fonknechten and Vincent Mottier for technical assistance and Jean-Yves Chatton and Yannick Krempp of the Cellular Imaging Facility (University of Lausanne) for experimental support. This research was supported by grants from the Swiss National Science Foundation (3100A0113925 and 310030-130769), from the Fondation Motrice, from the Société Académique Vaudoise, from the Fondation Marie-Thérèse et René Planiol and from the Fondation Emma Muschamp. Note

Supplemental materials can be found at: www.landesbioscience.com/journals/autophagy/article/16608 References


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